In a typical fluorescent imaging scenario for molecular pathology, dyes are used to label specific proteins or subcellular compartments. Often the proteins of greatest interest are expressed at low levels, and in the course of exciting the fluorescent reporters for imaging; the entire biological sample such as a tissue section is illuminated, generating fluorescence emission due to endogenous sources or non-specific binding outside of the targeted region-of-interest. The extra sources of emitted light are noise signals and typically must be removed or excluded by post-processing methods in order to accurately quantify to the low-expressing protein.
Structured Illumination Microscopy (SIM) is an imaging technique, which involves the use of such post-processing methods in which an illumination pattern or mask, is used to allow spatial control of the illumination and resolution of the specific cell region of interest. This method is used for such applications as autofluorescence reduction, extended dynamic-range imaging, and optical depth sectioning.
However, a need exists for a method, which can be used during the image acquisition itself to target specific regions of interest or to locally increase contrast and signal to noise ratio. A real-time SIM process would improve efficiency or specificity of the fluorescent excitation and analysis of samples using prior information or specific features in a tissue image to adjust subsequent illumination patterns. To accomplish this however, the process requires that the acquired image and the illumination mask be registered such that the illumination mask can be precisely superimposed on the sample during subsequent image acquisition.